Cell-support matrix having narrowly defined uniformly vertically and non-randomly organized porosity and pore density and a method for preparation thereof

ABSTRACT

A cell-support matrix having narrowly defined uniformly vertically and non-randomly organized porosity and pore density and a method for preparation thereof. The matrix suitable for preparation of cellular or acellular implants for growth and de novo formation of an articular hyaline-like cartilage. A gel-matrix composite system comprising collagen-based matrix having a narrowly defined porosity capable of inducing hyaline-like cartilage production from chondrocytes in vivo and in vitro.

This application is based on and claims priority of the provisionalapplication Ser. No. 60/718,714 filed on Sep. 19, 2005.

BACKGROUND OF THE INVENTION Field of Invention

The current invention concerns a biocompatible cell-support matrixhaving narrowly defined and uniformly vertically and non-randomlyorganized porosity and a pore density and a method for preparationthereof. In particular, the invention concerns a biocompatiblecell-support matrix or support substrate having substantially the samesize pores said matrix or substrate providing a support structure forlive-cell suspension. The matrix has vertically non-randomly orientedopen pores of substantially homogeneous pore size and narrowly defineddiameter.

The support matrix seeded with the chondrocyte suspension is suitablefor initiation of growth and de novo formation of hyaline orhyaline-like cartilage in vitro and in vivo as well as for preparationof cellular or acellular implants for implantation into articularcartilage in situ.

The invention additionally concerns a collagen-matrix composite systemcomprising of said support matrix seeded with chondrocyte suspension,said system being capable of induction of hyaline or hyaline-likecartilage from chondrocytes in vitro or in vivo when introduced into thearticular cartilage in situ.

BACKGROUND AND RELATED DISCLOSURES

Collagen matrices for use as an implant for repair of cartilage defectsand injuries are known in the art. Of particular interest is a honeycombstructure developed by Koken Company, Ltd., Tokyo, Japan, under thetrade name Honeycomb Sponge, described in the Japanese patent JP3170693.Other patents related to the current subject disclose collagen-basedsubstrate for tissue engineering (U.S. Pat. No. 6,790,454)collagen/polysaccharide bilayer matrix (U.S. Pat. No. 6,773,723),collagen/polysaccharide bilayer matrix (U.S. Pat. No. 6,896,904), matrixfor tissue engineering formed of hyaluronic acid and hydrolyzed collagen(U.S. Pat. No. 6,737,072), method for making a porous matrix particle(U.S. Pat. No. 5,629,191) method for making porous biodegradablepolymers (U.S. Pat. No. 6,673,286), process for growing tissue in amacroporous polymer scaffold (U.S. Pat. No. 6,875,442), method forpreserving porosity in porous materials (U.S. Pat. No. 4,522,753),method for preparation of collagen-glycosaminoglycan composite materials(U.S. Pat. No. 4,448,718), procedures for preparing composite materialsfrom collagen and glycosaminoglycan (U.S. Pat. No. 4,350,629) and acrosslinked collagen-mucopolysaccharide composite materials (U.S. Pat.No. 4,280,954).

However, many of the above disclosed structures have uncontrolledparameters such as uneven and uncontrolled porosity, uneven density ofpores, uneven sizes of the pores and random distribution of pores withinthe support matrix. Such uncontrolled parameters lead to structures thatare sterically unstable to provide support for cartilage matrixproducing cells as these structures easily collapse upon contact with asolution or suspension containing cartilage producing cells.

There is, therefore, a need for a more uniform and sterically stablesupport matrix preferably prepared from a biocompatible material, suchas collagen, wherein said matrix has narrowly defined size and densityof pores and the pores are uniformly distributed, vertically orientedand non-randomly organized.

It is, therefore, a primary object of this invention to provide a,sterically stable biocompatible, preferably collagen based matrix,having properties enabling chondrocyte attachment in numbers needed forinduction and formation of hyaline or hyaline-like cartilage.

The current invention provides such matrix and/or a method forfabrication thereof by providing a sterically stable and biocompatiblematrix, preferably made of Type I collagen, having narrowly defined poresizes and density with said pores organized vertically wherein saidmatrix permits seeding and attachment of chondrocytes suspended incollagen, gel, sol-gel or hydrogel that gels at the body temperature, insufficiently high numbers to induce formation of new hyaline orhyaline-like cartilage. The matrix according to the invention has asubstantially narrowly defined pore size in diameter and pore density invertically organized manner that creates an apical (top or synovial) orbasal (bottom or bone) surface to the implant where the sizes anddiameters of the pores on both the apical or basal surface aresubstantially the same. The gel system according to the inventionprovides conditions for a sterically-enhanced enablement of chondrocytesto produce extracellular matrix comprising glycosaminoglycan and Type IIcollagen and its deposition within said matrix in ratios characteristicfor normal healthy articular hyaline cartilage.

All patents, patent applications and publications cited herein arehereby incorporated by reference.

SUMMARY

One aspect of the current invention is a biocompatible support matrixhaving narrowly defined uniformly and vertically and non-randomlyorganized porosity and a pore density and a method for preparationthereof.

Another aspect of the current invention is a collagen-based supportmatrix having narrowly defined uniformly and vertically and non-randomlyorganized porosity and a pore density and a method for preparationthereof.

Another aspect of the current invention is a Type I collagen-basedsupport matrix suitable for seeding with chondrocytes suspension, saidmatrix having vertically oriented open pores of substantiallyhomogeneous pore size of narrowly defined diameter of about 200±100 μm.

Still another aspect of the current invention is the support matrixsuitable for growth and de novo formation of a hyaline-like cartilage invitro and for preparation of cellular or acellular implants for in situimplantation in vivo.

Yet another aspect of the current invention is a collagen-matrixcomposite system comprising a Type I collagen matrix and a suspension ofchondrocytes seeded into said matrix wherein said system is capable ofinduction of hyaline or hyaline-like cartilage from chondrocytes invitro or in vivo when said matrix is implanted with or without cells insitu.

Another aspect of the current invention is a method for preparation ofan uniform and sterically stable support matrix prepared from abiocompatible collagen material wherein said matrix has narrowly definedporosity and uniformly distributed vertically oriented and non-randomlyorganized pores of substantially the same size in diameter.

Still yet another aspect of the current invention is a method forpreparation of a sterically stable collagen-based matrix havingproperties, such as a pore size of about 200±100 μm in diameter, densityof about 25±10/mm², and uniform pore distribution enabling chondrocyteattachment in numbers needed for induction of formation of hyaline orhyaline-like cartilage.

Still yet another aspect of the current invention is a method forfabrication of a sterically stable type I collagen-based matrix thatpermits seeding of chondrocytes suspended in a Type-I collagen or in asynthetic sol-gel that gels at the body temperature, wherein said matrixhas a substantially narrowly defined pore size of about 200±100 μm and apore density 25±10/mm² in a vertically organized manner that assuresthat pores at a top (apical) and bottom (basal) surface of the implanthave substantially the same size.

Yet another aspect of the current invention is collagen, gel, sol-gel orhydrogel comprising a system that provides conditions for asterically-enhanced induction of chondrocytes into said matrix enablingsaid chondrocytes to produce extracellular matrix comprisingglycosaminoglycan and Type II collagen and its deposition within saidmatrix in the ratios characteristic for normal healthy articular hyalinecartilage.

Yet another aspect of the current invention is a method for producing aType I collagen-based matrix for seeding chondrocytes wherein saidmethod comprises preparation of said matrix from collagen suspension ofdefined collagen amount in the presence of ammonia, surfactant, atreduced pressure or in inert atmosphere or any combination of all theseparameters.

Still another aspect of the current invention is a gel-matrix compositesystem capable of inducing cartilage production comprising acollagen-based matrix seeded with a suspension of chondrocytes incollagen or a sol-gel polymer able to gel at a body temperature.

BRIEF DESCRIPTION OF FIGURES

FIG. 1A is a schematic side view of the collagen-based matrix having asubstantially homogeneous pore size of about 200±100 μm and narrowlydefined porosity with chondrocytes shown as if seeded in matrix pores ina substantially same distribution pattern within the matrix.

FIG. 1B is a schematic top view of a collagen-based matrix showing adistribution of as if seeded chondrocytes within the collagen-basedmatrix to be substantially homogeneous within the pores having a defineddiameter of about 200±100 μm.

FIG. 1C is a photograph of the actual Type I collagen matrix producedaccording to the method of the invention without magnification.

FIG. 1D is a photograph of approximately 2.5× magnified actual Type Icollagen matrix showing pore distribution within the matrix.

FIG. 1E is photograph of approximately 2.5× magnified actual Type Icollagen matrix with darkened background for better contrast showingpore distribution within the matrix.

FIG. 2 is a scheme of an experimental set-up for preparation of asupport matrix of the invention, providing means for storing andintroduction of aqueous ammonia, introduction of inert atmosphere andcreating reduced pressure.

FIG. 3A is a microphotograph of a Type I collagen-based matrix preparedfrom 4 mg/ml of collagen in the presence of 5 ml of 3% ammonia, showingthe pore size about 259±48 μm. The FIG. 3A clearly shows that the matrixhas pores of substantially the same size and an equal distributionthroughout the matrix. When the ammonia volume was increased to 6 ml,the pore size increased to 334±89 μm, as seen in FIG. 3B. Scale (1000μm, i.e. 1 cm) is shown in upper left corner.

FIG. 4A is a microphotograph of a Type I collagen-based matrix preparedfrom 6 mg/ml of collagen in the presence of 2 ml of 3% ammonia, showingthe pore size 233±55 μm, where the pore size and distribution of poresthrough the matrix is substantially the same. When under the sameconditions, the ammonia volume was increased to 4 ml, the pore sizeincreased only slightly to 235±67 μm, as seen in FIG. 4B. Scale (1000μm, i.e. 1 cm) is shown in upper left corner.

FIG. 5A is a microphotograph of a Type I collagen-based matrix preparedfrom 5 mg/ml of collagen in the presence of 1 ml of 3% ammonia preparedin an inert (nitrogen) atmosphere under reduced pressure (3 torr),showing the pore size 253±59 μm, wherein the pore size and distributionof pores through the matrix are substantially the same. When under thesame conditions, the nitrogen atmosphere pressure was further decreasedto 10 torr, the pore size increased to 323±82 μm, as seen in FIG. 5B.When under the same conditions the ammonia volume was increased to 3 ml,the size of pores increased to 538±135 μm, as seen in FIG. 5C. Scale(1000 μm, i.e. 1 cm) is shown in upper left corner.

FIG. 6A is a microphotograph of a Type I collagen-based matrix preparedfrom 4 mg/ml of collagen in the presence of 5 ml of 3% ammonia preparedin the presence of non-ionic surfactant (Pluronic® F68; 0.3% weight/%).The pore size at the surface of the matrix was 198±47 μm, showing thepore size and distribution of pores through the matrix to be homogeneousand the pore sizes substantially the same. When under the sameconditions, the amount of collagen was increased to 6 mg/ml, the poresize increased to 256±59 μm, as seen in FIG. 6B. When under the sameconditions the collagen was increased to 8 mg/ml, the size of pores roseto 380±100 μm, as seen in FIG. 6C. Scale is 500 μm shown in lower rightcorner.

FIG. 7A is a graph showing results of determination of a content ofS-GAG, measured by DMB assay, at day zero and day 21, obtained withseeding chondrocytes into the matrix having 200±50 pore sizes. FIG. 7Bis a graph showing a content of DNA measured by DNA assay at day zeroand day 21.

FIG. 8A is a graph showing a content of S-GAG produced by chondrocytesseeded in the support matrix having small pores (153±39 μm) or largepores (435±60 μm). FIG. 8B is a graph showing a content of DNA in thesmall and large pores, measured by DNA assay at day zero and day 21.

DEFINITIONS

“Sterically stable supporting structure” means non-random verticalorientation of collagen-fibrillar structure.

“Sterically unstable supporting structure” means random orientationmesh-like collagen-fibrillar structure.

“The matrix porosity” means a pore size defined by the diameter of holeswithin the support matrix or substrate as well as density of the poredistribution as a function of cross-sectional area in millimeters.

“Substantially homogeneous” means at least 85-99% homogeneity, that isat least 85% of all pores have sizes within the given range of 200±100μm. Preferable homogeneity is between 95 and 99%.

“Substantially homogeneous porosity” means that a pore size and diameteris within pore size range of about 200±100 μm, preferably 200±50 μm, indiameter.

“Top surface” means an apical or synovial side of the matrix turnedtoward the joint.

“Bottom surface” means basal, closest to bone surface of the matrix.

“Chondrocytes” means the cells naturally residing in articularcartilage.

“About 200±100 μm” means and is intended to include also pores where themean average is within additional 10-20 μm on the upper side.

“S-GAG” means sulfated glycosaminoglycan.

DETAILED DESCRIPTION OF THE INVENTION

The current invention is directed to a collagen-based matrix of definedporosity having substantially homogeneous pore sizes as well as to amethod for preparation thereof. The collagen-based matrix preparedaccording to the method of the invention has uniformly definedvertically non-randomly organized porosity and a defined pore density.

The support matrix or substrate is generally prepared from abiocompatible material such as collagen, particularly Type I collagen,and has vertically non-randomly oriented open pores of substantiallyhomogeneous pore size of a narrowly defined diameter. The matrix issuitable for preparation of acellular implants or cellular implantsseeded with chondrocytes, as well as for growth and de novo formation ofhyaline or hyaline-like cartilage in vitro and in vivo.

The invention additionally concerns a collagen-matrix composite systemcomprising said matrix seeded with a suspension of chondrocytes incollagen, gel, sol-gel or thermoreversible hydrogel, said system beingcapable of enabling formation of hyaline or hyaline-like cartilage bysupporting said chondrocytes in vitro or in vivo when said system isimplanted into damaged or injured cartilage in situ to produceextracellular matrix and its components Type II collagen and S-GAG.

The Type I collagen-based matrix of the invention has substantiallyhomogeneous pore size range of about 200±100 μm, preferably about 200±50μm, in diameter. This pore size has been shown to provide the largestattachment of the chondrocytes to the pores of the matrix.

The support matrix is ultimately useful for treatment of articularcartilage injuries and lesion by providing means for growing a newhyaline or hyaline-like cartilage for treatment, replacement orregeneration of the damaged or injured articular cartilage. Suchtreatment is currently difficult because of the unique properties of thearticular cartilage that is not the same as and does not behave as othersoft tissues.

I. Articular Cartilage Articular cartilage covers the ends of bones insynovial joints. Articular cartilage is an unique tissue in that is itis avascular, aneural and alymphatic and in mature state contains a verysmall number of cells. These properties are the main reason whyarticular cartilage has such a poor intrinsic capacity to heal (Install,J. N. and Scott, W. N., Surgery of the Knee, 3^(rd) Edition, p. 341(2001).

Articular cartilage is known as a relatively acellular tissue whoseextracellular space is occupied by interstitial fluid (60-80%) andorganic extracellular matrix (ECM) components, primarily proteoglycansand collagens.

Immature chondrocytes are the articular cartilage cells that are presentin large numbers in cartilage of young individuals. The immaturechondrocytes are metabolically active cells that are responsible forgrowth of cartilage in the young individuals. In adult individuals,where the growth of the bones has stopped, cartilage contains maturechondrocytes that are limited in number in mature quiescent cartilageand those present are mainly metabolically inactive.

The mechanical function of articular cartilage is determined by its highwater content and by the particular architecture of the collagennetwork. This network consists of cross-linked fibrils that extendperpendicular from the subchondral bone and curve gradually to a courseparallel to the articular surface in the superficial zone (“Form und Bauder Gelenkknorpel in ihren Beziehungen zur Funktion”, Z. Zellforsch.,Vol. 2, pp. 783-862 (1925)).

Any replacement support structure should thus have orientation andorganization similar to that observed naturally. Consequently, avertical orientation of a porous structure, preferably one made of abiodegradable material used in lieu of the cartilage matrix, would seemto be an important aspect for a cartilage regeneration in vivo due tothe similar structure of the native articular cartilage. Consequently, acombination of the vertical porous structure providing the necessarysupport combined with chondrocytes suspended in collagen, gel, sol-gelor another hydrogel would likewise seem to be beneficial for cartilagetreatment in vivo or regeneration of cartilage in vitro becausecollagen, gel, sol-gel or another hydrogel in which the chondrocytes aresuspended are biodegradable materials native or biocompatible witharticular cartilage and therefore can maintain chondrocytic phenotypeand stimulate cartilage extracellular matrix synthesis in vitro or in invivo when the support matrix seeded with chondrocytes is implanted insitu.

II. Collagen-Based Matrix and a Method for Preparation Thereof

The collagen-based matrix of the invention is an essential component ofa gel-matrix composite system capable of initiating the induction ofhyaline-like cartilage from chondrocytes.

A. Design of the Matrix

The matrix of the invention has been designed to meet requirements ofproperties needed for the support matrix.

The first requirement is that the support matrix is prepared from thebiocompatible and preferably biodegradable materials that are the sameor similar to those observed in the articular cartilage.

The second requirement is that the support matrix has a spatialorganization and orientation similar to that of the articular cartilage.

The third requirement is that the support matrix has a porositypermitting the seeding of the chondrocytes into said matrix in a numberof cells that are sufficient for initiation of a formation of newhyaline or hyaline-like articular cartilage in vitro and/or in vivo.

The fourth requirement is that the support matrix has sufficient numberof pores for the number of cells needed for initiation of articularcartilage formation and that the pore sizes are such that the majorityof chondrocytes seeded into said support matrix are suspended within thesupport matrix in numbers that would result in formation of new hyalineor hyaline-like cartilage.

The fifth requirement is that the pores have substantially the same sizein a range from about 200±100 μm, preferably between about 200±50 μm inpore diameter and that such size is substantially the same from the topapical to the bottom basal surface of the pores, said pores beingorganized vertically from the top to the bottom.

B. Support Matrix

Typically, the support matrix, preferably the collagen-based supportmatrix, of the invention is a three-dimensional structure made of abiocompatible and/or biodegradable material of defined density andporosity.

Typically, the support matrix is prepared from a collagenous gel or gelsolution containing Type I collagen, Type II collagen, Type IV collagen,gelatin, agarose, hyaluronin, a cell-contracted collagen containingproteoglycan, glycosaminoglycan or glycoprotein, fibronectin, laminin, abioactive peptide growth factor, cytokine, elastin, fibrin, a syntheticpolymeric fiber made of a poly-acid such as polylactic, polyglycolic orpolyamino acid, polycaprolactone, polyamino acid, polypeptide gel,copolymers thereof, each alone or in a combination. Additionally, thesupport matrix may be prepared from the collagen precursors. Forexample, the collagen precursors may be used to reconstitute collagenfibrillar structure for matrix protection. These precursors are, forexample peptide monomers, such as alpha 1 (type I), and alpha 2 (type I)collagen peptide or alpha 1 (type I) alpha 2 (type I) peptides, incombination, or 2 (alpha 1, type I) and 1 (alpha 2, type I) peptides.

Preferably, the support matrix of the invention is prepared fromcollagen and most preferably from Type I collagen, containing aplurality of narrowly defined uniformly vertically and non-randomlyorganized pores. The pores have substantially homogeneous narrowlydefined size and diameter and are uniformly distributed through thematrix dividing the matrix space into a fluid-filled column or network.

In preferred embodiments the Type I collagen-based matrix is acollagen-based sponge-like structure or honeycomb-like lattice ofdefined porosity having a vertically organized pores of substantiallysame sizes.

a. Defined Porosity

The support matrix of the invention has a certain thickness andvertically organized pores of a defined diameter oriented to create anapical (top or synovial) or basal (bottom or bone) surface of the matrixfor implantation. The diameter of said pores is chosen such that thematrix in conjunction with the chondrocyte-containing gel, preferably asol-gel, facilitates a sterically-enhanced induction of extracellularmatrix glycosaminoglycan and type II collagen deposition in ratioscharacteristic of hyaline articular cartilage.

b. Pore Sizes

The vertically oriented cellular or acellular support matrix made of thebiocompatible material having a predetermined pore size filled withchondrocytes suspension is important aspect of the quality of the repairtissue in vivo and for production of the articular cartilage in vitro.

Consequently, it is important to determine the optimal pore size for theporous matrix because the size of the pores of the matrix affects thechondrocyte attachment to the matrix walls and assures the presence ofchondrocytes within the matrix needed for cartilage regeneration invitro and in vivo. Sizes of the pores are substantially homogeneous withhomogeneity above 85%, preferably 95%, most preferably about 98-99% ofpores having a diameter size of about 200±100 μm, preferably 200±50 μm.

The pores of the collagen-based matrix are homogeneously distributedwithin said matrix to form a sponge-like structure able to taking in andevenly distributing the chondrocytes suspended in a gel solution andproviding conditions conducive to producing extracellular matrix by thesuspended chondrocytes.

The defined and substantially homogeneous pore size diameter of thecollagen-based matrix is an important aspect of the invention.Collagen-based matrices of defined pore sizes according to the inventionhaving different pore size diameter permit faster or slower infiltrationof the chondrocytes into said matrix, faster or slower growth andpropagation of the cells and, ultimately, the higher or lower density ofthe cells in gel-matrix system. Such pore size may be adjusted byvarying the pH of the gel solution, collagen concentration,lyophilization conditions, temperature, degree of cross-linking ofcollagen, etc.

Generally, in the prior art structures, the pore sizes are not definedand not limited to one substantially homogeneous size. thecollagen-based matrices known in the prior art are mostly structurescontaining pores having mixed pore sizes of from about 50 to about 2000μm.

The support matrix of the invention has the spatial organization andorientation similar to that of the articular cartilage. The spatialorganization and orientation of support matrix is schematically depictedin FIGS. 1A and 1B.

FIG. 1A is a schematic side view of the support matrix showing thevertical organization and orientation of support matrix pores seededhypothetically with chondrocytes suspended in sol-gel.

As seen in FIG. 1A, the pores are of substantially the same size indiameter and the number of cells in each pore is also substantially thesame. The cells shown here are round and touching the walls of the poresillustrating the attachment requirement for the support matrix.

Suspending solution for chondrocytes is any gel solution, preferably onecontaining collagen, gel, sol-gel or thermoreversible hydrogel that canchange its state from sol to gel depending on the temperature.

FIG. 1B is a schematic top view, showing hypothetically the ideallyhomogeneous distribution of pores through the support matrix, where thepores have the same sizes and chondrocytes are evenly distributed withinthe pores. The pores walls that separate individual pores are also seen.

FIG. 1B is a schematic top view of a gel-matrix composite systemcomprising the collagen-based matrix component made of Type I collagenand embedded with chondrocytes suspended in a sol-gel showing adistribution of chondrocytes within said collagen-based matrix to besubstantially homogeneous within the pores having a defined diameter ofabout 200 μm. The outer circle defines a size of the whole matrix wafer.Smaller circles are pores having a substantially the same size. Insideof the pore circles are chondrocytes deposited there in a sol-gelsolution.

FIG. 1C is the photograph of the actual Type I collagen matrix producedaccording to the method of the invention without magnification. Thematrix pores are visible as a black dots. The matrix can be cut to orderin both directions to obtain a perfect fit, if used, for example, for anarticular cartilage implant.

FIG. 1D is the photograph of 4× magnified actual Type I collagen matrixshowing the pore distribution within the matrix.

FIG. 1E is photograph of approximately 4× magnified actual Type Icollagen matrix with darkened background for better contrast showing thepore distribution within the matrix.

Chondrocytes suspended in a collagen, gel, sol-gel or thermoreversiblehydrogel solution are introduced into narrowly defined pores of asubstantially the same size and distribution within the matrix.Vertically separated columns have substantially the same diameter anddefined sizes of the pores.

The pores are filled with the chondrocyte suspension. This arrangementprovides sterically advantageous conditions for homogeneous distributionof chondrocyte deposition within the pores that lead to enhancedproduction of proteoglycans and type II collagen in ratios correspondingto the ratios observed for these compounds in the healthy hyalinecartilage.

The structural macromolecules and cartilage account for 20 to 30% of thewet weight of cartilage and includes type II collagen, large aggregatingproteoglycans and non-collagenous proteins or glycoproteins. Withmaturation, collagen contributes over 50% and proteoglycans contribute30-35% of the matrix dry weight.

The matrix is typically prepared as a composite cylindrical wafer seenin FIGS. 1C-1E or as a rectangular block structure cut into a wafer-likematrix construct having from about 4 mm to about 25 mm in diameter andthickness of from 0.5 to about 5 mm. The seeding density of thisconstruct is about 25,000-300,000 chondrocytes per 25 μl of collagensolution corresponding to about 1-12 millions cells/mL. The abovedensity numbers are exemplary only and even lesser seeding density ispossible and has been shown to lead to the production of the hyaline orhyaline-like cartilage. The cell density range for seeding is preferablyfrom about 1 to about 30 million/mL. Chondrocytes are preferablysuspended in a type I or Type II collagen or in a synthetic gel, sol-gelor hydrogel and such suspension, in combination with the support matrix,forms a gel-matrix composite system of the invention.

C. Preparation of the Support Matrix

The current invention additionally concerns a novel method forpreparation of the support matrix that meets the requirements stated insection IIA.

The method for preparation of the support matrix meeting such criteriainvolves process for standardizing the pore size, vertical orientationand organization, homogeneity and uniformity of the collagen-basedmatrix of the invention.

The fabrication method for preparation of the current support matrix isbased on investigation of various parameters comprising thebiocompatible components and/or additives and their amounts, or reactionconditions, such as, for example, pH, temperature, pressure, reducedpressure, presence of inert gasses and an apparatus for testing theseparameters all alone or in various combinations.

1. Apparatus for Preparation of the Support Matrix

Fabrication set-up for preparation of the support matrix of theinvention comprises variable components depending on the conditionsselected for the processing.

It typically comprises a sealed container containing the biocompatiblematerial or a precursor thereof, preferably Type I collagen, selectedfor the preparation of the support matrix wherein said material may besubjected to various conditions for testing and optimization or forfinal preparation of the support matrix. Added features may containmeans for increasing or decreasing pressure, temperature, adjusting pH,adding other components, pumps, gas tanks, valves, etc.

One illustrative set-up for preparation of the support matrix is shownin FIG. 2.

FIG. 2 is schematic representation of one experimental set-up used forpreparation of a support matrix of the invention, providing means forintroduction of aqueous ammonia, means for reducing pressure and meansfor providing an inert atmosphere. Typically, the starting material, inthis case the collagen solution, is placed into a sealed containerhaving an outlet connected to a vacuum gauge that is further connectedto a vacuum pump and a source of an inert gas. In FIG. 2, two sourcesare shown for supplying the aqueous ammonia and for storing nitrogenwith a means for introducing nitrogen into the system. The startingmaterial is then subjected to ammonia vapors by introducing apredetermined amount of aqueous ammonia into said container, and/orfurther introducing the inert gas, such as nitrogen or argon, or in onealternative, air, and optionally performing the reaction under reducedpressure, in the presence of a surfactant or under other reactionconditions. All these may be performed and/or tested in any combinationto achieve the optimal expected results.

2. Method for Preparation of the Support Matrix

The selected criteria for the pore size of the support matrix were setto prepare the support matrix having substantially homogeneouspopulation of pores within said matrix wherein said pores havesubstantially the same size in diameter of about 200±100 μm.

The preliminary studies determined that these parameters may be best metwith utilizing the Type I collagen in concentrations from about 2 toabout 10 mg/ml of collagen in solution, and these solutions were,therefore, further investigated for optimization of conditions forpreparation of the support matrix meeting the above set criteria.

The process for optimization of conditions for preparation of thesupport matrix meeting these criteria involved testing of Type Icollagen in 2, 4, 6, 8, and 10 mg per ml of solution, and itspolymerization in the presence of 3% aqueous ammonia added in 1, 2, 3,4, 5, and 6 ml volume. The same collagen preparations were tested withadded non-ionic surfactant (Pluronic® F86, 0.3 weight/%), and the samecollagen solutions were polymerized in an inert atmosphere and underreduced pressure. Various combinations of the above conditions werefurther tested to obtain the support matrix with the smallest and mosthomogeneous pore sizes.

Results are summarized in Tables 1-3 and in representative FIGS. 3-6.

a. Effect of the Ammonia Presence on Pore Sizes

In one embodiment, the support matrix was prepared from bovine Type Iatelocollagen (2.9 mg/ml, pH 2.1), obtained from Inamed Corporation,Fremont, Calif. The collagen was concentrated by a precipitationfollowed by centrifugation and then dissolved in aqueous HCl in order toreach the desired concentration. The procedure for preparation of thesupport matrix in the presence of ammonia is described in Example 3.

The pore sizes of the support matrix obtained under these conditions areexpressed as a function of collagen concentration and volume of ammoniasolution/concentration, is seen in Table 1.

TABLE 1 Collagen Volume of 3% aqueous ammonia conc. 2 ml 3 ml 4 ml 5 ml6 ml 2.8-2.9 mg/ml — — No pores — No Pores 4 mg/ml — 256 ± 68 274 ± 87259 ± 48* 334 ± 89 6 mg/ml 233 ± 55   365 ± 107  235 ± 67* — — 8 mg/ml284 ± 90*  299 ± 73 1195 ± 294 — — 10 mg/ml 474 ± 108* — Few — — pores

Pore size was measured within 1 mm of the surface of the formed matrix.

As seen in the Table 1, polymerization of collagen solution containingbetween 2.8 and 2.9 mg/ml did not result in a porous matrix formationregardless how much ammonia was added. Collagen at 4 mg/ml and at 3 mlor 5 ml ammonia polymerized into the matrix having a preferred and/oracceptable pore sizes in the range of 256±68 m and 259±48 μm,respectively. At 6 ml of ammonia, the matrix formed with pore sizes inthe range of 334±89 m.

FIG. 3A and FIG. 3B are representative microphotographs of supportmatrices prepared from 4 mg/ml collagen in the presence of 5 or 6 ml ofammonia resulting in pore sizes ranges of about 259±48 μm and about334±89 μm, respectively. Both microphotographs are on the same scale(scale 1000 μm) and the difference in their sizes is clearly visible.

Collagen in concentration of 6 mg/ml polymerized in the presence of 2 mlor 4 ml ammonia yielded the support matrix having pore sizes in theoptimal range of about 233±55 μm and about 235±67 μm, respectively. At 6ml of ammonia, the matrix formed with pore sizes in the range of 334±89μm.

FIG. 4A and FIG. 4B are representative microphotographs of supportmatrices prepared from 6 mg/ml collagen and 2 or 4 ml of ammoniaresulting in pore sizes ranges of about 233±55 μm and about 235±67 μm.Both microphotographs are on the same scale (scale 1000 μm). Bothmicrophotograps show the pores to be of approximately the same size.Homogeneity of their distribution is clearly visible.

Polymerization of collagen at 8 mg/ml collagen and at 2, 3, and 4 ml ofammonia resulted in the support matrix having larger pores in ranges284±90 μm, 299±73 μm and 1195±294 μm, respectively. At 10 mg/mlpolymerization yielded a matrix with pores in the range of 474±108 μmexemplifying inherent variability occurring with higher concentrationsof collagen.

As seen from these results, the support matrix prepared with a moderateamount of collagen between 4 and preferably 6 mg/ml, in the presence ofmoderate volume of ammonia, between 2 and 4 ml resulted in the matrixhaving the pore sizes within the optimal range of about 200±100 μm.

b. Effect of the Inert Atmosphere and Reduced Pressure

In another embodiment, the support matrix was prepared from bovine TypeI atelocollagen, as already described above and the support matrix wasprepared in the inert nitrogen atmosphere and at reduced pressure. Theprocedure conditions for preparation of the support matrix in thepresence of ammonia and at inert atmosphere and reduced pressure isdescribed in Example 5.

The support matrix obtained under these conditions and the pore sizesexpressed as a function of collagen concentration and volume of ammoniasolution/concentration are seen in Table 2.

TABLE 2 Volume 3%/NH3 Final pressure Pore size (μm) 1 ml 10 torr  323 ±82* 1 ml 3 torr 253 ± 59* 3 ml 10 torr  538 ± 135 6 ml 3 torr 557 ± 148

Table 2 shows the pore size as a function of volume of ammoniasolution/concentration and reduced pressure. Pore size is measuredwithin 1 mm of the surface of the formed scaffold.

As seen at Table 2, at a collagen concentration of 5 mg/ml in allinstances, and with presence of 1, 2 or 3 ml ammonia, the smallest poreswere obtained in an inert atmosphere combined with reduced pressure atthree torr. In that instance, the pore sizes were in the range from253±59 μm. All other combinations resulted in larger pore sizes above300 μm.

FIG. 5A shows the pore size of 253±59 μm of the matrix made of thepolymerization of 5 mg/ml collagen with 1 ml of ammonia in nitrogenatmosphere at reduced pressure to three torr.

FIG. 5B shows the matrix having a pore size of 323±82 μm made of thepolymerization of 5 mg/ml collagen with 1 ml of ammonia in nitrogenatmosphere at reduced pressure to 10 torr.

FIG. 5C shows the pore size of 538±135 μm of the matrix made bypolymerization of 5 mg/ml collagen with 3 ml of ammonia in nitrogenatmosphere at reduced pressure to 10 torr.

As seen from these results, the support matrix prepared with a smallvolume of ammonia combined with a moderate reduction of pressureperformed in an inert atmosphere resulted in the matrix having the poresizes within the optimal range of about 200±100 μm.

c. Effect of a Surfactant on Pore Sizes

In another embodiment, the support matrix was prepared from bovine TypeI atelocollagen, as described above. The procedure for preparation ofthe support matrix in the presence of surfactant is described in Example4.

The support matrix obtained under these conditions and the pore size isexpressed as a function of collagen concentration in the presence of the0.3% by weight of Pluronic® surfactant is seen in Table 3.

TABLE 3 Collagen Conc. Pores size (μm) 4 mg/ml 198 ± 47* 6 mg/ml 256 ±59* 8 mg/ml  380 ± 100*

Pore size seen in Table 3 is expressed as a function of collagenconcentration. Pore size is measured within 1 mm of the surface of theformed matrix.

As seen in Table 3, at a concentration of collagen at 4 mg/ml and in thepresence of the surfactant, the pore size of the support matrix wasabout 198±47 μm. At 6 mg/ml and at 8 mg/ml of collagen, the supportmatrix had pore sizes in the range from about 256±59 μm and about380±100 μm.

FIGS. 6A, 6B and 6C, having the same scale, are the microphotographs ofmatrices resulting from polymerization of 4, 6 and 8 mg/ml of collagenin the presence of the surfactant. These figures again clearly show theuniformity and homogeneity of the resulting pores as well as theiruniform sizes.

Presence of the surfactant in the collagen pre-polymerization mixtureclearly affects the formation of pore sizes within the optimal ranges,particularly when combined with lower concentration of collagen,polymerization of collagen (4 mg/ml) resulted in matrix having optimallysized pores of about 198±47 μm.

d. Other Factors

Some other factors may also positively affect the pore sizes similarlyto the above described ones.

For example, the collagen precursors may be used to reconstitutecollagen fibrillar structure for matrix protection. These precursorsare, for example peptide monomers, such as alpha 1 (type I), and alpha 2(type I) collagen peptides or in combination of 2 (alpha 1, type I) and1 (alpha 2, type I) peptides, or 3 (alpha I, type II) peptides.

The other factors that may be manipulated are the selective vorticalshearing of the peptide monomer, the chemical composition and pH of thesolubilization buffer.

The vortical shearing step determines the dispersion and alignment ofthe associated collagen fibrils of alpha I (type I) and alpha II (typeI) peptides. During this procedure equilibration in a nitrogen or argonatmosphere controls pH.

Other factors that may be used are the neutralization reactants forpolymerization, temperature, rate and percentage of water removal.

Exemplary conditions for preparation of the collagen-based matrix are asfollows.

The concentration of Type I collagen for fabrication of thecollagen-based matrix is about from about 2 to about 10, preferably fromabout 4 to about 8, most preferably about 4 to about 6 mg/ml. Suchconcentration of collagen is critical for the initial polymerizationresulting in fibrillogenesis, that is for formation of fibrils. Priorand current studies show that at the concentration of collagen below 3mg/ml there is no fibrillogenesis, and thus no pore formation, unlesssuch is promoted by addition of some other components.

The suitable buffers for solubilization of the Type I collagen are, forexample, a formic acid containing buffer at pH 4.8, acetic acidcontaining buffer at pH 5.0 or a diluted hydrochloric acid at pH 3.0.

The shearing of the collagen solution is set, for example, to be forabout 10-60 seconds at 10-100 dynes/cm².

The sheared peptides are equilibrated in an inert gas atmosphere such asnitrogen or argon gas for about 30 minutes to about 2 hours at about 4°C. The use of the inert gas displaces air containing oxygen that may actas an oxidant and decrease fibrillogenesis. This factor was found topositively affect the polymerization and pore formation.

Neutralization is typically carried out in a vapor of about 0.3% ammoniaover about 12 to about 24 hour period. This factor has also been foundto affect the collagen polymerization and formation of pores havinghomogeneous pore size.

Freezing, if there is, is carried out at −40° C. to about −60° C. over aperiod of about 2 to about 12 hours.

Water removal is achieved by exposure to an anhydrous atmosphere from−20° C. to about 50° C. preferably at 37° C. for about 24 to about 48hours.

The gradual nature of the polymerization and slow process of waterremoval typically maintains the architectural elements of the scaffoldcollagen-based to achieve the proper orientation and diameter of thelongitudinal pore structure necessary for hyaline cartilageextracellular matrix deposition by the cells.

The organization of the newly synthesized cartilage specific matrixwithin the porous type I collagen is visualized and quantified by, forexample, ELISA/Western blot methods for determination of protein levelsor quantitative RT-PCR or real-time PCR for m-RNA level.

III. Sterically-Enhanced Induction of Hyaline Cartilage

A main aspect of the current invention is a finding that when the poresizes are substantially homogeneously restricted to a narrowly defineddiameter, preferably to a pore size diameter of about 200±100,preferably 200±50 μm, and when such pores are vertically organized, suchdiameter of said pores in conjunction with the chondrocyte suspensionfacilitates a sterically-enhanced enablement of hyaline cartilageleading to formation of extracellular matrix. Such sterically-enhancedenablement leads to a deposition of collagen Type II and proteoglycanswithin the matrix in ratios characteristic of articular cartilage.

IV. Gel-Matrix Composite System Capable of Inducing Cartilage Production

In one embodiment, the invention concerns a collagen-based matrixprepared from Type I collagen seeded with chondrocytes suspended insynthetic sol-gel, which gels at body temperature.

The gel-matrix composite system comprises a matrix having pore diametersof about 200±100 μm, preferably 200±50 μm, that permit uniform infusionof chondrocyte/gel suspensions into the pores, and induces deposition ofcollagen Type II and proteoglycans (glycosaminoglycans) within thematrix in ratios characteristic of articular cartilage.

The collagen-based matrix of the current invention acts like a poroussponge when infiltrated with the suspended chondrocytes by, for example,wicking or infusion, wherein the cells are distributed within the matrixpores.

This arrangement permits chondrocytes to migrate and settle in thesupport matrix in a sterically-enhanced fashion and enables them toproliferate and secrete materials for generation of new extracellularmatrix and eventually producing a hyaline cartilage.

Suspending solution for chondrocytes is any gel solution, preferably onecontaining collagen, gel, sol-gel or theromoreversible hydrogel that canchange its state from sol to gel depending on the temperature, and ispreferably a thermo-reversible gelation hydrogel (TRGH) material inwhich the sol-gel transition occurs on the opposite temperature cycle ofagar and gelatin gels. Consequently, the viscous fluidic phase is in asol stage and the solid phase is in a gel stage. TRGH has very quicksol-gel transformation which requires no cure time and occurs simply asa function of temperature without hysteresis. The sol-gel transitiontemperature for embedding chondrocytes within the collagen-based matrixis set at a temperature where the sol-gel is in a sol state whereas thetemperature for stabilizing the chondrocytes within the matrix is set ata body temperature in the range of about 37° C.

V. Comparative Experimental Studies

Comparative experimental studies were performed in order tobiochemically evaluate a porous matrix seeded with a chondrocytesuspension in collagen and to further evaluate the effect of pore sizeof the porous support matrix on the chondrocytes proliferation andextracellular matrix formation.

These studies were performed using human chondrocytes. Healthy humanarticular cartilage (hAC) tissue was obtained from the Tissue Bank, TheNational Disease Research Interchange, Philadelphia, Pa. The cartilagetissue was digested, isolated chondrocytes were seeded on culture dishesand precultured for monolayer cell expansion. Isolated chondrocytes wereseeded in a 3D culture, according to a procedure described in Examples 7and 8.

A. Cell Seeding and Time-Dependent Production of Proteoglycan

For this study, the production of total sulfated glycosaminoglycan(S-GAG) content was measured by DMB assay after 0 and 21 days ofculture.

The experimental set-up is described in Example 6. Briefly, the cellswere seeded into the support matrix and cultured over night and theirS-GAG content and DNA (showing a number of cells) was determined at dayszero and 21.

Results are summarized in Table 4 and FIGS. 7A and 7B.

TABLE 4 Results of Biochemical Evaluation Biochemical Day 0 Day 21Results Mean SD Mean SD S-GAG 28.53 ±4.35 117.42* ±16.55 accumulationDNA contents 1.69 ±0.66 1.98 ±0.30 *P < 0.05

At day 0, the average of S-GAG content was 28.53±4.35 μg.

At day 21, the amount of S-GAG was 117.42±16.55 μg (Table 4 and FIG.7A). The S-GAG accumulation in the matrix at day 21 was significantlyincreased as compared to that at day 0. Results are seen in Table 4 andin FIG. 7A.

At day 21, the S-GAG accumulation was 4.2 fold greater than that at day0 (P<0.01). No statistical difference of the DNA content between day 0and 21 was observed, however tendency of increasing DNA content wasobserved at day 21 as compared to day 0.

FIG. 7A shows results of the DMB assay where the S-GAG content wasdetermined on day zero and day 21. As seen in FIG. 7A, culturing of thecomposite for 21 days led to a substantially increased production ofS-GAG. In contrast, the production of DNA did not increase under theseconditions, as seen in FIG. 7B, where DNA in g/composite was determined.

In the novel composite of collagen-based porous matrix comprisingchondrocytes suspended in a collagen hydrogel, chondrocyte proliferationand enhanced cartilage accumulation was observed. This finding indicatesthat the composite of the collagen-based porous matrix and hydrogelsuspension of chondrocytes is beneficial for cell adherence andproliferation and accumulation of the cartilage specific ExtracellularMatrix in vitro.

B. Evaluation of the Effect of Pore Size of the Collagen-Based Supporton Extracellular Matrix Production

For the evaluation of the effects of the pore size on biochemicalparameters, the total S-GAG content was measured by DMB assay after 21days of culture. Experimental procedure is described in Example 8.

The average of S-GAG content in constructs with a small pore size was183.01±39.78 μg (Table 5 and FIG. 8A). AS seen in Table 5 in FIG. 8A,the amount of S-GAG in constructs with a large pore size was115.56±13.50 μg (Table 4 and FIG. 8A) The S-GAG accumulation inconstructs with small pore size was significantly greater than that withlarge pore size (Table 5 and FIG. 8A).

The S-GAG accumulation in constructs with small pore size was 1.6-foldgreater than that with large pore size (Table 5 and FIG. 8A) (P<0.05).

The DNA content in constructs with a small pore size was significantlygreater than for constructs with a large pore size (Table 5 and FIG.8A). The DNA content in constructs with a small pore size was 1.5-foldgreater than that with a large pore size (Table 5 and FIG. 8A) (P<0.05).

TABLE 5 Results of Biochemical Evaluation Biochemical Small Pore LargePore Results Mean SD Mean SD S-GAG 183.01* ±39.78 115.56 ±13.50accumulation DNA Contents 2.78* ±0.21 1.81 ±0.10 *P < 0.05

Results of this study are illustrated in FIGS. 8A and 8B. FIG. 8A showsresults obtained in DMB assay where the S-GAG content per/composite isshown for a composite having large or small pores. As seen in FIG. 8A,small pores composites produced approximately one half more of S-GAGthan the composite having large pores. Similar results were seen in FIG.8B illustrating a DNA content in composite having a large or smallpores. As observed for S-GAG. The DNA content in the composite havingsmaller pores was significantly higher then in the composite having alarge pores.

As clearly seen in FIG. 8A, the production of S-GAG provides evidencethat the cartilage ECM formation was significantly higher in compositeshaving the small pores when compared to composites having the largepores. FIG. 8B is a graph showing a content of DNA, measured by DNAassay, in composites having small pores (153±39 m) or large pores(435±60 m). As clearly seen in FIG. 8B, the production of DNA wassignificantly higher in the composites having smaller pores as comparedto that with larger pores.

Small pore size of the composite porous matrix shows significant cellproliferation and cartilage specific accumulation in the matrix ascompared to that of large pore size.

C. Viability Determination

To establish biocompatibility of the support matrix of the invention,viability studies were performed. Experimental procedure is described inExample 9.

The matrices prepared with different collagen concentrations in thepresence of surfactant readily absorbed the chondrocyte-laden gel. Asseen in Table 6 below, the cell counts were consistent with the poresize distribution present in FIG. 6, with a cell viability of 98-99%.

TABLE 6 Mean % Group Mean cell count ± SD Viability Comments Table 6: 4mg/ml 100,800 ± 41,692 98.7 collagen with Pluronic Table 6: 6 mg/ml103,500 ± 15,679 99% collagen with Pluronic Table 6: 8 mg/ml 88,350 ±2,758 99.0 collagen with Pluronic

VI. Method for Use of Gel-Matrix Composite System

The matrix and a system of the invention are useful for production ofhyaline cartilage in situ or in vitro. In both cases, the collagen-basedmatrix is prepared as a matrix wafer.

For in situ use that is achieved by way of an implant, the matrix waferis cut into a size of the cartilage defect and introduced into thecartilage defect or lesion or the cartilage with some bone loss such asosteochondral defects.

The chondrocytes suspension is then introduced as a sol under colderthan body temperature into said matrix emplaced in the lesion or defectthereby generating a gel-matrix composite system in situ and thetemperature is raised to the body temperature whereby the sol istransitionally changed into a gel.

In alternative, the invention works in the same way for acellularimplant where the collagen-based matrix filled with the sol-gel isintroduced without chondrocytes. The implant is left in the body untilthe new hyaline cartilage is generated and the matrix which isbiodegradable self-degrades.

For in vitro use, the process is similar but proceeds in the tube orPetri dish under the same conditions until the hyaline cartilage isproduced. Such cartilage may then be used as an implant into thecartilage defect or lesion.

Example 1

Preparation of the Collagen-Based Matrix

This example describes one exemplary method for preparation of thecollagen-based matrix.

Type I collagen is dissolved in a formic acid buffer at pH 4.8 and itsconcentration is adjusted to about 5.2 mg/ml. The solution is subjectedto a vortical shearing for 10 seconds at 10 dynes per cm. The shearedpeptides is then equilibrated in nitrogen gas for 30 minutes at 4° C. todisplace air. Neutralization is carried out in a vapor of 0.3% ammoniaover a 24 hour period. The solution is then subjected to freezing at−40° C. over a period of 2 hours. Water is removed by exposing thefrozen solution to an anhydrous atmosphere at 37° C. for 24 hours.

The organization of the newly synthesized cartilage specific matrixwithin the porous type I collagen is visualized and quantified usingimmunohistochemical methods and matrix-specific gene expressionquantified by in situ mRNA hybridization.

Example 2 Preparation of Collagen-Based Matrix

This example illustrates another exemplary method for preparation of thecollagen-based matrix.

300 grams of a 1% aqueous atelocollagen solution (VITROGEN®), maintainedat pH 3.0, is poured into a 10×20 cm tray. This tray is then placed in a5 liter container. A 50 mL open container containing 30 mL of a 3%aqueous ammonia solution is then placed next to the tray, in the 5 literchamber, containing 300 grams of said 1% aqueous solution ofatelocollagen. The 5 liter container containing the open trays ofatelocollagen and ammonia is then sealed and left to stand at roomtemperature for 12 hours. During this period the ammonia gas, releasedfrom the open container of aqueous ammonia and confined within thesealed 5 liter container, is reacted with the aqueous atelocollagenresulting in gelling said aqueous solution of atelocollagen.

The collagenous gel is then washed with water overnight and,subsequently, freeze-dried to yield a sponge like matrix.

This freeze dried matrix is then cut into squares, sterilized, andstored under a sterile wrap.

Example 3 Effect of Ammonia on Preparation of Porous Honeycomb Scaffold

This example illustrates a general procedure utilizing ammonia forpreparation of porous honeycomb scaffold having substantially the samesize and distribution of pores.

About 30 g collagen solution (concentrations listed in table 1) with apH of 3.0-4.8 was added to a 100 ml glass beaker. The solution wascentrifuged for 5-10 minutes at 800×g to remove air bubbles. Aftercentrifugation the beaker with collagen solution was sealed in a 7.1 dm³container together with 3% aqueous ammonia. The collagen solution wasprecipitated in the presence of ammonia gas for 3-14 h, forming verticalcone shaped pores where the diameter increased with the depth. Afterprecipitation the collagen gel was washed with deionized water for 1-3days in order to remove excess ammonia and formed salts. The washedcollagen was then slowly frozen and lyophilized.

Example 4 Effect of Surfactant on Pore Size

This example illustrates preparation of porous honeycomb scaffold in thepresence of a surfactant.

20 g of collagen solution with 0.3 weight % of Pluronic® F68 (BASF), anon ionic surfactant with a pH of 3.5-3.8, was added to a 100 ml glassbeaker. The collagen concentration versus the final pore size is listedin the table below. The solution was centrifuged for 5-10 minutes at800×g to remove air bubbles. After centrifugation the beaker withcollagen solution was sealed in a 7.1 dm³ container together with 3%aqueous ammonia. The collagen solution was precipitated in the presenceof ammonia gas for 2 h, forming vertical cone shaped pores where thediameter increased with the depth. After precipitation the collagen gelwas washed with deionized water for 1 day in order to remove excessammonia and formed salts. The washed collagen was then slowly frozen andlyophilized.

Example 5 Effect of Inert Atmosphere and Reduced Pressure on Pore Size

This example illustrates preparation of honeycomb porous scaffold ininert atmosphere (N₂) and under reduced pressure. 15 g of bovine type 1atelocollagen dissolved in aqueous HCl with a concentration of 5 mg/mland pH of 3.3 was added to a 100 ml glass beaker. The solution wascentrifuged for 5-10 minutes at 800×g to remove air bubbles. Aftercentrifugation the collagen solution was placed in 9.1 dm³ container.The container was sealed and the air evacuated using a vacuum pump to apressure of about 2 torr (water almost boiled). The pump was turned offand the system was filled with nitrogen to about 27 torr. Evacuation andfilling was repeated three times. Before the addition of ammonia thepressure was again reduced to about 2 torr and the vacuum pump and flaskwere closed off. Aqueous ammonia was charged to the flask and after 30seconds the connection to the container was opened and nitrogen was usedto flush the ammonia gas into the container with the collagen solution.The final pressure in the container was in the range of 3-15 torr. Theprecipitation and formation of pores was complete in 40 minutes.

The formed pores were vertical and cone shaped and the diameterincreased with increasing distance from the surface of the collagen gel.The precipitated collagen was washed with deionized water for 1 day inorder to remove excess ammonia and formed salts. The washed collagen wasthen slowly frozen and lyophilized.

Example 6 Evaluation of Time-Dependent Production of Proteoglycan

This example describes a study performed for evaluation of theimportance of the pore size for induction of production of a hyalinearticulate cartilage.

To evaluate the effect of the pore size of through porous matrix, 2different pore sized matrices were prepared. Large pore matrix had poresof an average size of 435 μm. Small pore matrix had pores of an averagesize of 153 μm.

Cells were harvested with trypsin-EDTA (Invitrogen). Three hundredthousand chondrocytes obtained from human articulate cartilage (hACs)were suspended in hydrogel (collagen gel) and seeded into a composite ofa porous sponge matrix having an average pore size 435 m or an averagepore size 153 μm. The composites were then transferred into the culturemedium.

After 12 hours pre-incubation, cell constructs were cultured in mediumcomprising 5% CO₂, 2% O₂ and 37° C. in a multigas incubator usingDMEM/F-12 medium with 10% FBS, 1% ITS, 0.1% gentamycin (Invitrogen).After 21 days of culture, constructs were harvested for biochemicalevaluation.

Example 7 Biochemical Evaluations

This example describes methods used for biochemical evaluation ofcollagen-based composites.

For biochemical analysis, composites were digested in papain at 60° C.for 18 hours. DNA was measured using the Hoechst 33258 dye methoddescribed in Anal. Biochem., 174:168-176 (1988).Sulfated-glycosaminoglycan (S-GAG) content was measured using a modifieddimethylmethylene blue (DMB) microassay described in Connect. TissueRes., 9:247-248 (1982).

Example 8 Cell Seeding in a Three-Dimensional Collagen-Based Matrix

This example describes the procedure used for cell harvesting andseeding in the collagen-based matrix according to the invention.

Cells were harvested with trypsin-EDTA (Invitrogen). Three hundredthousand human articular chondrocytes (hACs) were suspended in acollagen hydrogel and seeded into the composite of porous collagen-basedmatrix having a predetermined average pore size 435 μm or 153 μm. Thecell constructs were incubated at 37° C. for about one hour collagengelation and then transferred into the culture medium. After 12 hourspre-incubation, the composites comprising chondrocytes were cultivated.After zero and 21 days of culture, constructs were harvested forbiochemical evaluation.

Example 9 Viability Determination

This example illustrates determination of cell viability.

Collagen-based matrices prepared by the surfactant method were seededwith approximately 200,000 chondrocytes in a collagen gel by absorptionand incubated for 3 days. An n=3 was used for each group. Attermination, the chondrocyte-contained matrices were placed in 1.5 mlmicrocentrifuge tubes and incubated overnight in 0.15% collagenase. Thedigest was spun at 2000 rpm for 5 minutes and the supernatant aspirated.An aliquot of culture medium (0.1 ml) was added to the cell pellets andan aliquot taken for counting. Cell viability and total cell count wasmeasured using trypan blue.

1. An implant comprising: a porous body comprising collagen, wherein the body has a top surface, an opposed basal surface, and a plurality of pores of substantially uniform pore size and orientation extending from the top surface to the basal surface.
 2. The implant of claim 1, wherein about 95% of the pores have a diameter of about 200 plus or minus 100 microns.
 3. The implant of claim 1, wherein about 95% of the pores have a diameter of about 200 plus or minus 50 microns.
 4. The implant of claim 1, wherein about 98% of the pores have a diameter of about 200 plus or minus 100 microns.
 5. The implant of claim 1, wherein about 98% of the pores have a diameter of about 200 plus or minus 50 microns.
 6. The implant of claim 1, wherein the collagen comprises one selected from the group consisting of type I collagen, type II collagen, and type IV collagen.
 7. The implant of claim 1, wherein the collagen is type I collagen.
 8. The implant of claim 1, further comprising a gel disposed within the pores.
 9. The implant of claim 8, wherein the gel is a sol-gel.
 10. The implant of claim 9, wherein the sol-gel has a sol-to-gel transition temperature of about 37 degrees C.
 11. The implant of claim 8, wherein the gel comprises a plurality of chondrocytes suspended therein.
 12. The implant of claim 11, wherein the chondrocytes include human articular chondrocytes.
 13. The implant of claim 8, wherein the gel comprises a collagen solution.
 14. The implant of claim 11, wherein the porous body and the gel are in the form of a wafer-like construct having a diameter from about 4 mm to about 25 mm and a thickness from about 0.5 mm to about 5 mm.
 15. The implant of claim 14, wherein the wafer-like construct has been cut from a composition cylindrical or rectangular block structure.
 16. The implant of claim 8, wherein the gel further includes a plurality of chondrocytes suspended therein.
 17. The implant of claim 16, wherein the gel is a sol-gel that gels at a body temperature.
 18. The implant of claim 16, wherein the chondrocytes are suspended within the collagen solution at a seeding density of about 75,000-300,000 chondrocytes per 25 microliters of the collagen solution.
 19. The implant of claim 14, wherein the sol-gel is a collagen solution comprising type I collagen.
 20. A cartilage implant comprising: a porous body comprising type I collagen disposed between a top surface and an opposed basal surface of the porous body, the porous body having a plurality of pores of substantially uniform pore size and orientation extending from the top surface to the basal surface, wherein about 95% of the pores have a diameter of about 200 plus or minus 100 microns; a sol-gel that gels at a body temperature disposed within the pores; and a plurality of chondrocytes suspended within the sol-gel.
 21. The implant of claim 20, wherein: the chondrocytes include human articular chondrocytes; the porous body and the gel are in the form of a wafer-like construct having a diameter from about 4 mm to about 25 mm and a thickness from about 0.5 mm to about 5 mm; the sol-gel is a collagen solution comprising type I collagen; and the chondrocytes are suspended within the collagen solution at a seeding density of about 75,000-300,000 chondrocytes per 25 microliters of the collagen solution. 